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目的:探索针对诱导性组织因子(TF)高表达的靶向抑制的方法,探讨针对TF启动子-247～-230区域的诱骗寡核苷酸对U937-PR9细胞中PML/RARα融合蛋白诱导的TF表达是否存在竞争性抑制作用。方法:采用诱骗寡核苷酸技术,模拟TF启动子-247～-230的序列,人工合成该片段的双链DNA,转染U937-PR9细胞,通过流式细胞术监测转染效率并确定最佳电转浓度,通过实时PCR和酶联免疫吸附试验等技术,比较转染前后加Zn2+组与不加Zn2+组的TF表达水平变化。结果:流式细胞术显示,10μmol/L电转浓度下能够实现高效转染,标记的寡核苷酸在细胞内能够稳定存在72 h。未转染对照组中,加Zn2+诱导后TF表达明显上升;转染组中,加Zn2+诱导的TF表达水平与不加Zn2+组几乎没有差异。结论:特异性诱骗寡核苷酸对TF诱导性高表达存在靶向的竞争性抑制作用,该方法为急性早幼粒细胞白血病TF高表达引发的并发症提供了靶向治疗的新思路。
OBJECTIVE: To explore a method of targeting inhibition of induced high expression of tissue factor (TF), and to explore the effect of decoy oligonucleotide directed against TF promoter -247 ~ -230 on PML / RARα fusion protein in U937-PR9 cells Whether there is competitive inhibition of TF expression. Methods: The decoy oligonucleotide was used to simulate the sequence of -247 ~ -230 in TF promoter. The double-stranded DNA of this fragment was synthesized and transfected into U937-PR9 cells. The transfection efficiency was determined by flow cytometry Good electroporation concentration, real-time PCR and enzyme-linked immunosorbent assay and other techniques, compared with before and after transfection plus Zn2 + group with or without Zn2 group TF expression levels. Results: Flow cytometry showed that the transfected cells could efficiently transfect at the concentration of 10μmol / L, and the labeled oligonucleotides could stably exist in cells for 72 hours. In the untransfected control group, the expression of TF increased obviously with the addition of Zn2 +, and there was almost no difference between the transfected group and the Zn2 + -induced TF expression level. CONCLUSIONS: Targeted competitive inhibition of TF-induced high expression by specific decoy oligonucleotides provides a novel strategy for targeted therapy of complications associated with high TF expression in acute promyelocytic leukemia.